VAAM-Jahrestagung 2012 18.–21. März in Tübingen

145system. Even though, eukaryotes obtain TrxR system as well, the similarityis very low, therefore TrxR systems can be targeted to treat tuberculosis.After an in silico high throughput screening for Trx-inhibitors, fourdifferent low mass scaffolds were identified. In vitro testing of compoundsrelying on those scaffolds was performed at recombinant expressed MtbTrxR. The auspicious substances were tested in liquid Mtb cultures withMGIT 960 system (Becton Dickinson). We identified several substancesthat showed bacteriostatic effects on Mtb at M concentrations.We could show that by attacking the TrxR-system in vitro mycobacterialgrowth can be arrested.OTP033Nitrous oxide reductase with a unique [4Cu:2S] centre fromdenitrifying Pseudomonas stutzeriA. Wüst* 1 , L. Schneider 1 , A. Pomowski 1 , W.G. Zumft 2 , P.M.H. Kroneck 3 ,O. Einsle 11 Albert-Ludwigs-Universität Freiburg, Institut für Organische Chemie undBiochemie, Freiburg, Germany2 Karlsruher Institut für Technologie, Molekulare Mikrobiologie, Karlsruhe,Germany3 Universität Konstanz , Dept. of Biology, Konstanz, GermanyThe genera Pseudomonas and Paracoccus include the most commonlyisolated denitrifying bacteria from soils and aquatic sediments and mayrepresent the most active denitrifiers in natural environments 3 .Denitrification is the dissimilatory reduction from the ionic oxides (nitrateand nitrite) to the gaseous oxides nitric oxide and nitrous oxide. Thesubsequent two-electron reduction of nitrous oxide to dinitrogen is thefinal step in the denitrification process 4 . Nitrous oxide is involved inatmospheric reactions and its accumulation in the stratosphere leads todepletion of ozone.Nitrous oxide reductase, NosZ, is a dimeric multi-copper protein, with 638residues per subunit (74 kDa) and the reported copper content depends onthe purification strategy. Because of the high sensitivity of the enzymetoward dioxygen, the clusters of the soluble periplasmic enzyme degradeand it therefore loses its activity under aerobic conditions. In literature,several different forms were described, that can be distinguished by theirtypical absorption and EPR spectra. The active purple form of the enzymecarries the well-characterized mixed-valent binuclear Cu A centre and thetetranuclear Cu Z site, that was first described as a unique [4Cu:2S] centre 1for Pseudomonas stutzeri, instead of the [4Cu:S] cluster 4 found previously.This newly described cluster was observed after the isolation andcrystallization under the exclusion of dioxygen 2 . In nitrous oxide reductasethe substrate N 2O is bound between the two copper centres, it is activatedby side-on binding at Cu Z, so that then electrons can be transferred directlyfrom Cu A to N 2O. Several accessory proteins were identified for thebiogenesis of active N 2O reductase, with predicted functions as Cuchaperones or ABC transporters. To date the exact steps of clusterbiogenesis and the mechanistic details of N 2O reduction are still unknown.OTP034Key enzymes of fuel oxygenate ether degradationJ. Schuster*, F. Schäfer, N. Yaneva, T. Rohwerder, R.H. Müller, H. HarmsHelmholtz Centre for Environmental Research GmbH - UFZ , Departmentof Environmental Microbiology , Leipzig, GermanyThe extensive use of methyl-tert-butyl and tert-amyl methyl ether (MTBEand TAME, respectively) as gasoline additives has resulted in persistentgroundwater contamination due to their recalcitrance against microbialattack. However, we were able to isolate the bacterial strain Aquincolatertiaricarbonis L108 from an MTBE-contaminated aquifer (Leuna,Germany) which can grow well on all kinds of fuel oxygenate ethers assingle source of carbon and energy [1]. We have now elucidated theunderlying degradation pathways by generating gene knockoutsspecifically affecting expression of key enzymatic steps. In addition,central metabolites of ether catabolism were identified. Initial degradationproceeds via specific hydroxylation by the EthABCD monooxygenasesystem resulting in the formation of tert-butyl or tert-amyl alcohol (TBAor TAA). Degradation of the latter is mainly catalyzed by themonooxygenase MdpJ. TBA is hydroxylated to 2-methylpropan-1,2-diol,while TAA is desaturated to the hemiterpene 2-methyl-3-buten-2-ol. In aside reaction, TBA and TAA are dehydrated to the corresponding alkenes,i. e. isobutene and isoamylene isomers, by a not yet characterizedenzymatic step [2]. The 2-methylpropan-1,2-diol is oxidized further to 2-hydroxyisobutyric acid, which is activated to the corresponding CoA esterand isomerized to the common metabolite 3-hydroxybutyryl-CoA by aspecific cobalamin-dependent acyl-CoA mutase [1]. 2-methyl-3-buten-2-ol, on the other hand, is degraded via a hemiterpenic primary alcohol andthe corresponding aldehyde and carboxylic acid, linking TAA degradationwith the biotin-dependent catabolism of the amino acid leucine [3].T. Rohwerder, U. Breuer, D. Benndorf, U. Lechner and R.H. Müller, Appl. Environ. Microbiol.72 (2006), p.4128.F. Schäfer, L. Muzica, J. Schuster, N. Treuter, M. Rosell, H. Harms, R.H. Müller and T. Rohwerder, Appl.Environ. Microbiol.77 (2011), p. 5981.We kindly acknowledge DBU (Deutsche Bundesstiftung Umwelt) for financial support of F. Schäfer (AZ:20008/994) and C. Schumann (UFZ) and M. Neytschev (UFZ) for technical assistance and B. Wuerz (UFZ)for excellent analytical advice.OTP035Marinobacter adhaerens hp15 is required for aggregation ofthe diatom, Thalassiosira weissflogiiM. Ullrich*, A. Gaerdes, E. Sonnenschein, S. Seebah, I. Torres-MonroyJacobs University Bremen, Molecular Life Science Research Center,Bremen, GermanyAggregation of diatoms is an important process in marine ecosystemsleading to the settling of particulate organic carbon predominantly in theform of marine snow. Exudation products of phytoplankton formtransparent exopolymer particles (TEP), which act as adhesives for particleaggregation. Heterotrophic bacteria interacting with phytoplankton mayinfluence TEP formation and phytoplankton aggregation. This bacterialimpact has not been explored in detail. We hypothesized that bacteriaattaching to Thalassiosira weissflogii might interact in a yet-to-bedetermined manner, which could impact TEP formation and aggregateabundance. The role of individual T. weissflogii-attaching and free-livingnew bacterial isolates for TEP production and diatom aggregation wasinvestigated in vitro. T. weissflogii did not aggregate in axenic culture, andstriking differences in aggregation dynamics and TEP abundance wereobserved when diatom cultures were inoculated with either diatomattaching,i.e. Marinobacter adhaerens HP15, or free-living bacteria. Thedata indicated that free-living bacteria may not influence aggregationwhereas bacteria such as M. adhaerens HP15 may increase aggregateformation. Interestingly, photosynthetically inactivated T. weissflogii cellsdid not aggregate regardless of the presence of bacteria. Comparison ofaggregate formation, TEP production, aggregate sinking velocity, and solidhydrated density revealed remarkable differences. Both, photosyntheticallyactive T. weissflogii and specific diatom-attaching bacteria were requiredfor aggregation. It was concluded that interactions between heterotrophicbacteria and diatoms increased aggregate formation and particle sinkingand thus may enhance the efficiency of the biological pump. M. adhaerensHP15 has become a genetically accessible model organism. Successfulsite-directed and transposon mutageneses, expression or reporter genes,and full access to the genome sequence of HP15 made this organism anideal model strain to conduct the molecular dissection of the diatombacteriainteraction at the cell-to-cell level.OTP036Will be presented as OTV032!OTP037Carbon stable-isotope fractionation of brominated ethenes bySulfurospirillum multivoransA. Woods*, I. NijenhuisHelmholtz-Centre for Environmental Research-UFZ, IsotopeBiogeochemistry, Leipzig, GermanyMicrobial dehalogenation has been investigated as a viable remediationstrategy for contaminated field sites, as several bacterial species have beenlinked with biotransformation processes including reductivedehalogenation. Compound specific isotope analysis (CSIA) may be aneffective tool for monitoring reductive dehalogenation activity in theenvironment if distinct fractionation patterns emerge duringbiodegradation studies in the laboratory. While the biodegradationpotential of chlorinated ethenes has been extensively characterized, verylittle is known as regards biotransformation of brominated ethenes.However, certain bacterial strains, including Sulfurospirillum multivoransand Desulfitobacterium sp. strain PCE-S, which are capable of reductivedechlorination of tetrachloroethene (PCE) and trichloroethene (TCE) to1,2-dichloroethene (1,2-DCE), have also been shown to effectivelydebrominate tribromoethene (TBE) and 1,2-dibromoethene (1,2-DBE)under similar conditions [1].Carbon stable-isotope fractionation had previously been determined duringreductive dechlorination of PCE and TCE by S. multivorans andDesulfitobacterium sp. strain PCE-S [2], but had not been tested for thecorresponding brominated compounds. This study aims to investigate thecarbon-isotope fractionation of TBE and 1,2-DBE during reductivedebromination by crude extracts of S. multivorans, and to evaluate theseresults against those for their chlorinated analogs. Preliminary results showbrominated ethene fractionation patterns as similar to those for chlorinatedethenes by each strain, but to a lesser extent. In the case of TBE,fractionation was nearly negligible, contrasting with significantfractionation observed for TCE. However, fractionation observed duringreductive debromination of 1,2-DBE, while less than that observed for 1,2-DCE, is significant, and suggests the potential use of CSIA for in situassessments of reductive debromination. To further explore this potential,more studies are required to investigate fractionation occurring with otherstrains and likewise, with chlorinated ethenes.BIOspektrum | Tagungsband 2012